The present invention relates to a novel freeze-drying (lyophilization) method.
Freeze-drying is an important method for stabilizing hydrolysis-sensitive and thermolabile preparations, and of materials of biological origin which are to be dried under gentle conditions. Using freeze-drying, materials can be dried without relatively great changes or losses of biological activity. A beneficial aspect of freeze-drying is that the dried, xe2x80x9clyophilicxe2x80x9d products, owing to their porous structure and very high specific surface area, can be very rapidly reconstituted and regain their original properties in solution. Therefore, freeze-drying is preferably used for therapeutic sera, blood products, biologically active substances (hormones, vitamins, enzymes, medicaments), food preparations and flavorings. Suitable preparations for freeze-drying are liquid and semi-solid aqueous preparations, for example solutions, emulsions and suspensions.
Drying from the frozen state combines the advantages of freezing and dehydration at low temperature and is generally carried out in the following manner:
cooling and crystallization of the solvent in the preparation at atmospheric pressure.
main drying, that is to say sublimation of the crystallized solvent.
further drying, that is to say evaporation of noncrystallized solvent fractions.
The two drying steps differ in principle: during the main drying (primary drying) the frozen solvent is sublimated under reduced pressure. During the optional further drying (secondary drying) nonfrozen solvent evaporates at reduced pressure and at elevated temperature.
In the methods known from the prior art, the preparations to be dried are frozen in vessels, termed vials, at atmospheric pressure and the product temperature is set to a value suitable for starting the main drying.
Freezing (crystallization) is followed by the main drying, during which at reduced pressure the frozen solvent is converted from the solid to the gaseous aggregate state, that is to say is sublimated. The energy which is consumed during sublimation is supplied, for example, via heatable adjustable shelves. During the main drying the frozen preparation must not heat up above its melting point. The main drying can be followed by further drying, in which the nonfrozen solvent is removed at elevated temperature and reduced pressure. This involves solvent which can be, for example, adsorbed on the solid matrix, or enclosed in amorphous areas. Crystallization in the present application is taken to mean freezing (solidification) the solvent in the preparation. Preparation in the present application is taken to mean any type of material which is suitable for freeze-drying.
The temperature course during freeze-drying can be controlled by suitable apparatuses. Those which are known to those skilled in the art are, in particular, thermostatable adjustable shelves. The adjustable shelves can, in this method, be brought to the desired freezing temperature both after loading (cooling variant A) and before loading (cooling variant B). It is also possible to precool the plates and/or the preparation on the plates to a temperature above the actual freezing temperature in order to ensure temperature uniformity of the individual vials or to minimize the cooling time before freezing. This is followed by the actual freezing with further lowering of the shelf temperature (cooling variant C). Variants A-C describe freezing on adjustable shelves. Other known methods are freezing methods in cooling baths and rotating vessels (shell freezing, spin freezing) or by spray apparatuses; they differ in principle from the methods described above. Usually, the preparations to be dried are aqueous systems. In principle, other solvents or their mixtures with aqueous systems can also be used, for example carboxylic acids (for example glacial acetic acid), dimethyl sulfoxide (DMSO), ether (for example dioxane), dimethylformamide or alcohols (for example t-butanol).
The various conventional types of freezing and of freeze-drying are adequately described, for example in relevant text books, for example Lyophilization, Essig, Oschmann, Wissenschaftliche Verlagsgesellschaft Stuttgart mbH, 1993; pages 15-29, Gefriertrocknen [freeze-drying], Georg-Wilhelm Oetjen, VCH Verlag, 1997; pages 3-58, and Freeze Drying, Athanasios I. Liapis, in: Handbook of Industrial Drying, ed. by A. S. Mujumdar, Montreal, page 295-326.
All freezing methods have in common the fact that, if the preparation is suitable, after the freezing a tempering step (thermal treatment or annealing) can be performed. This tempering step serves to promote the crystallization of amorphously solidified solids and nonfrozen solvents and thus to achieve an increased crystallinity and reduced residual moisture. To carry it out, the frozen preparation is heated to a temperature which is above the glass transition temperature (Tgxe2x80x2) of the amorphously solidified solution and is below the melting point of the solution. The amorphous phase, which generally has high contents of noncrystallized solvent, is converted from the glass state to the rubberlike state and the mobility of molecules is increased. The consequence is the formation of nucleoli that grow to form crystals (eruptive recrystallization) and the addition of solvent molecules to pre-existing solvent crystals.
The tempering method is also known in the literature. Descriptions of the tempering method may be found in The Lyophilization of Pharmaceuticals: A Literature Review, N. A. Williams and G. P. Polli, Journal of Parenteral Science and Technology, (March-April 1984) 38 (2) 48-59, Basic Aspects and Future Trends in the Freeze-Drying of Pharmaceuticals, L. Rey, Develop. biol. Standart., Vol. 74, (Karger, Basel, 1991), pp. 3-8 und Fundamental Aspects of Lyophilization, L. Rey, Researches and Development in Freeze-Drying, ed. by L. Rey, Paris, 1964, 24-47.
The lyophilizates produced using the freeze-drying methods of the prior art mostly have a high resistance to flow, which hinders the escape of gaseous solvent. In addition, the dissolved constituents may not crystallize out completely or at all, so that products are obtained which are partly to completely amorphous. The consequences which can result from this are mechanical damage of the product cake due to the escaping solvent vapor stream and as a result potential loss of product, and collapsing and thawing phenomena during drying. Furthermore, the end user also imposes esthetic requirements in particular on pharmaceutical and food preparations, so that severe damage is not desired.
An object of the present invention was therefore to find a freeze-drying method using which lyophilizates may be produced which do not have the abovementioned problematic properties and are therefore easier to handle.
Surprisingly, it has now been found that lyophilizates which are more mechanically stable are obtained if the freeze-drying method is carried out as follows:
Phase 1: Reducing the pressure in the drying chamber until the onset of a visible crystallization of the solvent at a temperature in the drying chamber which is above the solidification point of the preparation.
Phase 2: Reduction of the temperature in the drying chamber to a temperature which is below the solidification point of the preparation or is identical to this, until completion of crystallization of the solvent.
Phase 3: Sublimation of the frozen solvent by means of reduced pressure.
By the solidification point of the preparation there is meant in the present application the temperature at which the solvent in the preparation is transformed into the solid aggregate state.
According to the invention the pressure in the drying chamber at the start, with a temperature in the drying chamber which is above the solidification point of the preparation, is reduced to a pressure below atmospheric pressure (according to FIG. 1). This causes a surface cooling of the preparation by evaporation and partial crystallization of the solvent on the surface (phase 1). In a preferred embodiment the pressure in this case with aqueous solutions is 0.1 to 6 mbar, in particular 0.2 to 3 mbar. This pressure p in the drying chamber (measured using a capacity manometer) is plotted for various preparations as a function of the concentration c (in mol/L) in FIG. 1. The values for various aqueous preparations was shown as follows:
continuous line, squares=mannitol
continuous line, circles=sucrose
continuous line, lozenges=sodium chloride
dashed line, circles=glycine
dashed line, triangles=maltose
square on the y-axis=solvent water
This pressure reduction can be performed, for example, at room temperature. In a further embodiment the preparations, before or during the pressure reduction, are precooled to a temperature which is between room temperature and the solidification point of the preparation. This precooling (for example on adjustable shelves) further ensures that the cooling apparatuses which sometimes have low cooling rates, can be brought in a short time to the desired crystallization temperature, that is to say in the region of the solidification point of the preparation. It is critical that this precooling does not lead to crystallization of the solvent.
If crystals have formed, for example in the form of a water/ice mixture or an ice layer floating on the surface, the pressure in the drying chamber can be raised again to ambient pressure and the temperature in the drying chamber for crystallization can be brought to or below the solidification point of the preparation (phase 2). It is also possible to keep the pressure reduced during the crystallization; this has no relevant effects on the solvent crystallization. In principle, for the crystallization, any temperature is suitable which is below the solidification point of the preparation or is identical to it. In a preferred embodiment, the temperature for the crystallization in the case of aqueous solutions is between xe2x88x9260xc2x0 C. and 0xc2x0 C.
After crystallization, the preparation, if appropriate, is brought to the final temperature for the start of drying. This temperature depends on the product present and, via the vapor pressure curve of the solvent, on the pressure which is to be used in the primary drying. In a preferred embodiment this temperature in the case of aqueous solutions is xe2x88x9260xc2x0 C. to 0xc2x0 C.
The primary drying then follows. This proceeds in principle as in the methods according to the prior art. In a further embodiment the method additionally has a secondary drying phase (phase 4) after the primary drying. However, in the event of a tempering phase (phase 2a), this is not necessary in some cases.
According to a further embodiment, a tempering method as described above follows phase 2. This tempering method is designated below as phase 2a. Tempering gives products with higher crystallinity and lower residual moisture after the primary drying and shortens the secondary drying or makes it superfluous.